Therapeutic use of p75ntr neurotrophin binding protein

ABSTRACT

The present invention relates to a p75NTR neurotrophin binding protein, p75NTR(NBP), for use in the treatment of pain and/or a symptom of pain.

BACKGROUND TO THE INVENTION

The neurotrophins, neurotrophic growth factor (NGF), brain-derivedneurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5(NT-4/5) act via four receptors: the low affinity p75 neutrophicreceptor (p75NTR), and the high affinity tyrosine kinase receptors;TrkA, TrkB, and TrkC. The low affinity receptor p75NTR binds and isactivated by all four neurotrophins and has been reported to functionindependently from the other receptors. However, the Trk receptors aremore selectively activated i.e. NGF is the selective ligand for TrkA,BDNF the ligand for TrkB and NT-3, 4/5 the ligands for TrkC. In additionit has been reported, when p75NTR and Trk proteins are co-expressed,they form complexes, which alter the signaling of both receptors (Huangand Reichardt, 2003). Indeed, it has been suggested that p75NTRfacilitates the selectivity of each of the neutrophins for theirrespective Trk receptor.

The p75NTR is a member of the tumor necrosis factor receptor superfamily(TNFR-SF) and was the first member of this superfamily to becharacterized fully. The superfamily (encoded by some 30 genes inhumans) is defined by ligand-binding domains consisting of one or more(typically four) repeats of a 40 amino acid cysteine-rich domain (CRD)that was first identified in p75NTR (Johnson et al., 1986; Radeke etal., 1987). In contrast, no sequence motif is shared by theintracellular domains of all TNFR-SF family members. Consequently,signaling mechanisms of TNFR-SF proteins vary significantly.

An unusual feature of p75NTR structure is the existence of adisulfide-linked p75NTR dimer, formed via cysteinyl residues within thetransmembrane domains. This disulfide linkage is required for effectiveneurotrophin-dependent signaling by p75NTR and plays an important rolein the formation of an intracellular and extracellular domain (Vilar etal., 2009b). Neurotrophins exist physiologically as noncovalentlyassociated dimers (Bothwell and Shooter, 1977) with a distributionhalf-life of approximately 5 min (Tria et al., 1994).Neurotrophin-dependent p75NTR activation involves association of aneurotrophin dimer with CRDs 2-4 of the two extracellular domains of ap75NTR dimer (He and Garcia, 2004). Recent studies support a model inwhich neurotrophin binding causes the two extracellular domains ofp75NTR dimers to move closer together, forcing the intracellular domainsto splay apart in a snail-tong-like motion centered on the disulfidebond and permitting association of the intracellular domains with thesignaling adapter proteins, NRIF and TRAF6 (Vilar et al., 2009a, 2009b).Intra-transmembrane domain disulfide bonds, such as are present inp75NTR, have not been described previously in other TNFR-SF familymembers, or in any other membrane protein.

p75NTR undergoes sequential proteolytic cleavage by α-secretase andγ-secretase activities and matrix metalloproteinases (MMPs), releasingits intracellular domain (ICD) into the cytoplasm, in a manner analogousto the cleavage-dependent signaling pathway of Notch and β-amyloidprecursor protein (Jung et al., 2003; Kanning et al., 2003). Cytoplasmicrelease of the p75NTR ICD by this pathway promotes signaling byassociated NRIF (Kenchappa et al., 2006). The role of the extracellulardomain of p75NTR, following the proteolytic cleavage by α-secretase andγ-secretase activities and MMPs isn't fully understood.

It has been documented that NGF and other neurotrophins (BDNF, NT-3 andNT-4/5) play a significant role in pathology for example pain due toosteoarthritis, pancreatitis, rheumatoid arthritis, psoriasis, pruritisand multiple sclerosis (Watanabe et al., 2010; Raychaudhuri et al.,2011; Barthel et al., 2009; Truzzi et al., 2011; McDonald et al., 2011;Yamaoka et al., 2007). It was been demonstrated that selectiveantibodies to any of the neutrophins; either NGF or BDNF, NT-3 andNT-4/5 significantly reduce pain. Furthermore, antibodies directed tothe neurotrophin receptors p75NTR Trk A, Trk B or Trk C have also beendemonstrated to be efficacious in models of pain (Orita S et al., 2010;Svensson P et al., 2010; Iwakura et al., 2010; Cirilio et al., 2010;Pezet et al., 2010; Hayashi et al., 2011; Chu et al., 2011; Ueda et al.,2010; Ghilardi et al., 2010; Fukui et al., 2010). Fukui et al., (2010)in a model of pain (mechanical allodynia following sciatic nerve crush)demonstrated significant efficacy on pain related endpoints followingtreatment with an anti-p75NTR antibody. It was concluded from this studythat the treatment with a p75NTR inhibitory antibody reduced CGRP andp75NTR expression resulting in a significant reduction in pain.

The current invention demonstrates the extracellular domain of p75NTR,exogenous or endogenous following cleavage from the cell membrane, actsas a neurotrophin binding protein or soluble receptor to each of theneurotrophins NGF, BDNF, NT-3 and NT-4/5 and plays a significant role inthe function, physiology and homeostasis of neurotrophin function.Moreover, we describe the use of the extracellular domain of p75NTR formodulating or neutralizing the pathological actions of the neurotrophicfactors including NGF, BDNF, NT-3 and NT4/5, for example in models ofstatic allodynia and thermal hyperalgesia. Thus the p75NTR neurotrophinbinding protein finds use in the treatment of pain and otherneurotrophic factor related pathologies such as psoriasis, eczema,rheumatoid arthritis, cystitis, endometriosis and osteoarthritis.

BRIEF DESCRIPTION OF THE INVENTION

According to a first aspect of the present invention there is provided ap75NTR neurotrophin binding protein, (NBP), for use in the treatment ofpain, or for the prevention and/or treatment of pain and/or symptoms ofpain or for ameliorating, controlling, reducing incidence of, ordelaying the development or progression of pain and/or symptoms of pain.According to a second aspect of the present invention there is provideda p75NTR neurotrophin binding protein for use according to the firstaspect wherein, the p75NTR(NBP) comprises p75NTR(NBP) connected to oneor more auxiliary molecules. Preferably, the p75NTR(NBP) is connected tothe one or more auxiliary molecules via one or more linkers. Accordingto a third aspect of the invention there is provided a nucleic acidmolecule encoding the p75NTR(NBP) or p75NTR(NBP) connected to one ormore auxiliary molecules of the p75NTR(NBP) according to the first orsecond aspects the nucleic acid molecule may further comprise encoding asignal sequence. Also provided is the use of the nucleic acid moleculefor the treatment of pain. According to a fourth aspect of the inventionthere is provided a replicable expression vector for transfecting acell, the vector comprising the nucleic acid molecule of the thirdaspect, preferably the vector is a viral vector. Also provided is theuse of the vector for the treatment of pain. According to a fifth aspectof the invention there is provided a host cell harbouring the nucleicacid molecule of either the third or fourth aspect. According to a sixthaspect of the invention there is provided the p75NTR(NBP) for useaccording to the first or second aspect or the preferred embodimentsthereof, or the nucleic acid or vector for use according to the thirdand fourth aspects wherein the p75NTR(NBP) or the nucleic acid moleculeor vector is for separate, sequential or simultaneous use in acombination combined with a second pharmacologically active compound.

DESCRIPTION OF THE FIGURES

FIG. 1. p75NTR extracellular domain sequence, neurotrophin bindingdomains highlighted in bold.

FIG. 2a and FIG. 2b . p75NBPP peptide standard fragments andco-immunoprecipitation of p75NTR(NBP) and NGF demonstratingp75NTR(NBP)-NGF complex in human plasma. FIG. 2a shows detection ofp75NTR extracellular domain standard. FIG. 2b shows detection of p75extracellular domain from total NGF assay using commercial human serum.

FIG. 3. MS/MS sequence confirmation of the WADAECEEIPGR (SEQ ID NO. 7)peak shown in FIGS. 2a and 2 b.

FIG. 4. p75NTR(NBP) inhibits NGF function in U20S cell line expressingTrkA (top panel) and in U20S cell line co-expressing TrkA and p75NTR(bottom panel).

FIG. 5. Soluble p75NTR(NBP) inhibits BDNF function in U20S cell lineexpressing TrkA (top panel) and in U20S cell line co-expressing TrkA andp75NTR (bottom panel).

FIG. 6. Biocore data demonstrating p75NTR(NBP) binding to BDNF andcompetition between anti-BDNF and p75NTR(NBP).

FIG. 7. p75NTR(NBP) inhibition of NGF activity in PC12 cells. Top showssignificant inhibition of activity in cultured cells. Bottom showschange in cell number for different concentrations of p75NTR(NBP).

FIG. 8. Neurotrophin Binding Protein p75NTR(NBP) inhibits BDNF paineffect in a model of nerve excitability. Top half of figure shows before(upper) and 45 minutes after (lower) administration of BDNF,demonstrating a significant increase in neuronal excitability. Bottomhalf of figure demonstrates that the addition of p75NTR(NBP) to themedia significantly reduced the neuronal excitability in response topinching stimuli.

FIG. 9. Neurotrophin Binding Protein p75NTR(NBP) inhibits hyperalgesiain UVIH model

FIG. 10. SEQ ID NO. 1 Human p75NTR full amino acid sequence

FIG. 11. SEQ ID NO. 2 Human p75NTR extracellular domain including signalsequence

FIG. 12. SEQ ID NO. 3 Human p75NTR extracellular domain without signalsequence

FIG. 13. SEQ ID NO. 4 Human p75NTR(NBP) neurotrophin binding domain 1

FIG. 14. SEQ ID NO. 5 Human p75NTR(NBP) neurotrophin binding domain 2

FIG. 15. SEQ ID NO. 6 Human p75NTR(NBP) neurotrophin binding domain 3

FIG. 16. SEQ ID NO. 7 Human p75NTR(NBP) neurotrophin binding domain 4

FIG. 17. SEQ ID NO. 8 Human p75NTR(NBP) neurotrophin binding domain 5

FIG. 18. SEQ ID NO. 9 Human Transferrin

FIG. 19. SEQ ID NO. 10 Human Albumin

FIG. 20. SEQ ID NO. 11 Human Fc IgG1

FIG. 21. SEQ ID NO. 12 Human Fc IgG2

FIG. 22. SEQ ID NO. 13 Human Fc IgG3

FIG. 23. SEQ ID NO. 14 Human Fc IgG4

FIG. 24. SEQ ID NO. 15 Human Pc Fragment Engineered For Extended SerumHalf-Life

FIG. 25. SEQ ID NO. 16 Human Fc Fragment Engineered For Lack Of EffectorFunctions

FIG. 26. SEQ ID NO 17 p75NTR(NBP)-Fc linker

FIG. 27: SEQ ID NO. 18 p75NTR(NBP)-Fc linker

FIG. 28: SEQ ID NO. 19 p75NTR(NBP)-Fc linker

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the present invention there is provided ap75NTR neurotrophin binding protein, (p75NTR(NBP)), for use in thetreatment of pain, or for the prevention and/or treatment of pain and/orsymptoms of pain or for ameliorating, controlling, reducing incidenceof, or delaying the development or progression of pain and/or symptomsof pain. Preferably the p75NTR neurotrophin binding protein,p75NTR(NBP), is pegylated, further preferably it is glycosylated.

The p75NTR neurotrophin binding protein, p75NTR(NBP), preferablycomprises (a) one or more of neurotrophin binding domain 1 [SEQ ID NO.4], 2 [SEQ ID NO. 5], 3 [SEQ ID NO. 6], 4 [SEQ ID NO. 7] or 5 [SEQ IDNO. 8]. Further preferably the p75NTR(NBP) comprises (b) each ofneurotrophin binding domain 1 [SEQ ID NO. 4], 2 [SEQ ID NO. 5], 4 [SEQID NO. 7] and 5 [SEQ ID NO. 8] or (c) each of neurotrophin bindingdomain 1 [SEQ ID NO. 4], 3 [SEQ ID NO. 6], 4 [SEQ ID NO. 7] and 5 [SEQID NO. 8]. Further preferably the p75NTR(NBP) comprises extracellulardomain 2 [SEQ ID NO. 3] or a portion thereof comprising (a), (b) or (c)above. Further preferably the p75NTR(NBP) comprises extracellular domain1 [SEQ ID NO. 2] or a portion thereof comprising (a), (b) or (c) above.

Preferably the p75NTR(NBP) binds to each of the neurotrophins NGF, NT3,BDNF and NT4/5, preferably human NGF, NT3, BDNF and NT4/5. Furtherpreferably the p75NTR(NBP) binds to each of the neurotrophins NGF, NT3,BDNF and NT4/5, preferably human NGF, NT3, BDNF and NT4/5, preferablywith a binding constant equivalent to or the same as the native completesequence p75NTR [SEQ ID NO. 1]. Binding constants can be determinedusing the assays described herein such as by use of surface plasmonresonance at 20° C., assays for the native protein binding constants areknown and comprise cell based assays known in the art. Preferably thep75NTR(NBP) protects one or more of the aforementioned neurotrophinsfrom degradation in plasma or other bodily fluids, in-vitro or in-vivo,and/or maintain a homeostatic balance of one or more of theaforementioned neurotrophins compared to their biologically free form.

The p75NTR(NBP) of the present invention preferably binds to any one ormore of NGF, BDNF, NT3 or NT4/5 with a binding affinity (K_(d)) ofbetween about 0.1 nM to about 50 nM. In some preferred embodiments, thebinding affinity (K_(d)) is between about 0.1 nM and any of about 0.2nM, 0.5 nM, 1 nM, 1.5 nM, 2 nM, 2.5 nM, 3 nM, 3.5 nM, 4 nM, 4.5 nM, 5nM, 5.5 nM, 6 nM, 6.5 nM, 7 nM, 7.5 nM, 8 nM, 8.5 nM, 9 nM, 9.5 nM, 10nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM or 50 nM as measuredin an in vitro binding assay for NGF, BDNF, NT3 or NT4/5 such asdescribed herein preferably as measured by surface plasmon resonance at20° C. In some further preferred embodiments, binding affinity (K_(d))is or is less than any of about 250 pM, 300 pM, 350 pM, 400 pM, 450 pM,500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 950 pMor 1 nM as measured in an in vitro binding assay for p75NTR(NBP) withthe neurotrophins such as described herein, preferably as measured bysurface plasmon resonance at 20° C. In a further more preferredembodiment the binding affinity (K_(d)) is about 0.3 nM or about 1 nM,as measured in an in vitro binding assay for p75NTR(NBP) with theneurotrophins such as described herein, preferably as measured bysurface plasmon resonance at 20° C.

Further preferably the p75NTR(NBP) effects the functional activity ofthe aforementioned neurotrophins, (defined as modulating or up or downregulating the functional activity of the neurotrophins) NGF, BDNF, NT3or NT4/5, for example the functional activity of the aforementionedneurotrophins resulting from their interaction with their respectivereceptors.

Preferably the p75NTR(NBP) effects the functional activity of BDNF asassessed by functional assay of any of growth and differentiation ofneurons and synapses, survival and differentiation in neuronal cellculture, Trk signalling, stimulation of axon outgrowth in vitro or invivo.

Preferably the p75NTR(NBP) effects the functional activity of NGF asassessed by measuring NGF binding to and activation of TrkA, asdemonstrated in classical neuron survival assays (such as provided inCowan, W. M., Hamburger, V., Levi-Montalcini, R. Annu. Rev. Neurosci.2001; 24:551-600).

Preferably the p75NTR(NBP) effects the functional activity of NT3 asassessed by measuring NT3 binding to and activation of endogenous Trkreceptor activity, as demonstrated in Trk receptor phosphorylation,mitogen-activated protein kinase phosphorylation reporter assays or cellsurvival and neurite extension assays.

Preferably the p75NTR(NBP) effects the functional activity of NT4/5 asassessed by measuring NT4/5 in vitro or in vivo phosphorylation andactivation assays for example in myelin basic protein (MBP)phosphorylation assays or alternatively in vivo in a Matrigelangiogenesis assay of vascular endothelial growth factor (VEGF)/basicfibroblast growth factor-induced angiogenesis.

Preferably the p75NTR(NBP) binds to the contact residues of one or moreof the neurotrophins NGF, NT3, BDNF and NT4/5 as shown in He and Garcia(2001) Science, 301, pages 870-805.

Preferably the p75NTR(NBP) is soluble, preferably soluble in aqueoussolution, preferably soluble in a biological fluid such as serum,plasma, blood.

According to a second aspect of the present invention there is provideda p75NTR(NBP) for use according to the first aspect wherein, thep75NTR(NBP) comprises p75NTR(NBP), preferably the p75NTR(NBP) of thefirst aspect, connected to one or more auxiliary molecules. Preferably,the p75NTR(NBP) is connected to the one or more auxiliary molecules viaone or more linkers.

Preferably the one or more auxiliary molecules are selected from; (a)transferrin or a portion thereof, preferably the transferrin is humantransferrin, preferably SEQ ID NO. 9, (b) albumin or a portion thereof,preferably the albumin is human albumin, preferably SEQ ID NO. 10 (c) animmunoglobulin Fc or a portion thereof, preferably the immunoglobulin Fcis human immunoglobulin Fc, (d) a polyethylene glycol polymer chain (e)a carbohydrate chain.

As used herein, the term, “immunoglobulin Fc” or “Ig Fc” is understoodto mean the carboxyl-terminal portion of an immunoglobulin chainconstant region, preferably an immunoglobulin heavy chain constantregion, or a portion thereof. Preferably the immunoglobulin Fccomprises 1) a CH1 domain, a CH2 domain, and a CH3 domain, optionallywith an immunoglobulin hinge region, 2) a CH1 domain and a CH2 domain,optionally with an immunoglobulin hinge region, 3) a CH1 domain and aCH3 domain, optionally with an immunoglobulin hinge region, 4) a CH2domain and a CH3 domain, optionally with an immunoglobulin hinge regionor 5) a combination of two or more domains selected from CH1, CH2 andCH3 optionally combined with an immunoglobulin hinge region. Preferablythe immunoglobulin Fc comprises at least an immunoglobulin hinge region,a CH2 domain and a CH3 domain, and optionally a CH1 domain. Preferablythe immunoglobulin Fc comprises or consists of an Fc or a portion of anFc of an immunoglobulin of isotype selected from IgG, IgM, IgA, IgD,IgE, further preferably, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, sIgA, morepreferably IgG2 or IgG4, most preferably IgG2. Optionally theimmunoglobulin Fc also comprises amino acid mutations, deletions,substitutions or chemical modifications which serve to minimisecomplement fixation or antibody-dependent cellular cytotoxicity or whichimprove affinity of binding to the Fc receptor.

Further preferably the immunoglobulin Fc comprises or consists of anyof: (a) a CH2 domain or portion thereof and a CH3 domain or portionthereof, (b) a CH2 domain or portion thereof, or (c) a CH3 domain orportion thereof, wherein the immunoglobulin Fc or portion thereof is ofisotype selected from IgG, IgM, IgA, IgD, IgE, further preferably, IgG1,IgG2, IgG3, IgG4, IgA1, IgA2, sIgA, more preferably IgG2 or IgG4, mostpreferably IgG2.

Preferably the immunoglobulin Fc comprises or consists of the carboxyterminal region of an immunoglobulin heavy chain and may comprise theCH2 and/or CH3 domains, or parts thereof, from IgG, IgA or IgD antibodyisotypes, or the CH2 and/or CH3 and/or CH4 domains, or parts thereoffrom IgM or IgE. Preferably the immunoglobulin Fc comprises or consistsof a fragment of the Fc, comprising mainly CH3 and a small portion ofCH2, as is derivable by pepsin digestion of the immunoglobulin.Preferably the immunoglobulin Fc comprises or consists of the full Fcregion, comprising CH2 and CH3, additionally connected to the hingeregion which is a short segment of heavy chain connecting the CH1 andCH2 regions in the intact immunoglobulin, as may be produced by papaindigestion of the immunoglobulin. Preferably the immunoglobulin hingeregion comprises or consists of a hinge region or part of a hinge regionderived from an IgG preferably human IgG, more preferably selected fromIgG1, IgG2, IgG3, or IgG4, most preferably IgG1 or is alternatively aspecies or allelic variant of the foregoing hinge region embodiments.The hinge region or a part of an immunoglobulin hinge region can belocated at the C or N-terminal end of the Fc region, preferably at theN-terminal end.

According to a preferred embodiment of the present invention theimmunoglobulin Fc preferably comprises or consists of an Fc or a portionof an Fc of an immunoglobulin which comprises one or more amino acidmutations of the wild type sequence in the CH2 region which reduce Fceffector function. Preferably these mutations are A330, P331 to 5330,5331 (amino acid numbering with reference to the wildtype IgG2 sequence,wherein the CH2 region is in the human heavy chain IgG2 constant region:[Eur. J. Immunol. (1999) 29:2613-2624]. Preferably the immunoglobulin Fcis glycosylated and highly charged at physiological pH hence helpingsolubilise the p75NTR(NBP). The Fc region also permits detection of thep75NTR(NBP) by anti-Fc ELISA for example in diagnostic purposes. Thep75NTR(NBP) of the invention is preferably synthesized in a cell whichglycosylates the Ig Fc preferably at normal glycosylation sites.

Preferably the immunoglobulin Fc comprises or consists of a humanimmunoglobulin Fc region of amino acid sequence selected from SEQ ID No.11, 12, 13, 14, 15 or 16 or a species or allelic variant thereof, or theCH2 and or CH3 domains, or portions thereof derived from SEQ ID NO. 11,12, 13, 14, 15 or 16.

According to the present invention, the p75NTR(NBP) connected to one ormore auxiliary molecules preferably demonstrates advantageous biologicalproperties of improved solubility of p75NTR(NBP) and/or stability ofp75NTR(NBP) and/or improved serum half life p75NTR(NBP). Improvedsolubility is desirable in order that bioavailability of the p75NTR(NBP)is maximized on administration and accurate dosage of the p75NTR(NBP)can be determined and carried out. Improved solubility is advantageousto overcome the problem of aggregates which are undesirable causing painin delivery in-vivo and leading to potential inflammation. Improvedserum half life has the advantage of facilitating reduced levels orreduced frequency of dose requirement during use for treatment in orderto achieve the equivalent or maintained therapeutic effect of thep75NTR(NBP) delivered. A prolonged half life and higher stability inblood or serum has the advantage of permitting a dosage regime of lessfrequent dosing and/or lower dosing levels hence reducing potentialtoxicity or side effects in-vivo. In this case the p75NTR(NBP) is morepotent in its therapeutic effect and/or more stable in the circulation.The resulting lower or less frequent doses are advantageous inminimising any potential toxic effects or side effects potentiallyassociated with p75NTR(NBP) administration. The molecular weight of thep75NTR(NBP) connected to one or more auxiliary molecules is alsoincreased over p75NTR(NBP) alone, this has the advantage that themolecule will be well retained in the blood circulation whenadministered intravenously reducing the risk of penetration to undesiredsites for example the central nervous system and making the moleculesuitable for retention or concentration in the tissues targeted.

Preferably the p75NTR(NBP) connected to one or more auxiliary moleculesdemonstrates improved solubility of p75NTR(NBP) and/or improvedstability of p75NTR(NBP) and/or improved serum half life in comparisonto p75NTR(NBP) not so connected. Preferably the improved solubility issolubility in an aqueous solution such as water preferably withexcipients such as buffers and/or salts at preferably at a physiologicalpH, preferably at between pH 5 to pH 8, preferably about pH 7, or issolubility in a biological fluid such as serum or blood. Preferably theimproved stability is stability of activity or structural integrity ofthe p75NTR(NBP) protein due to the effects of denaturation, oxidation,fragmentation or aggregation over a period of time, during a periodstorage or following freeze and thaw. Structural stability can be judgedby standard measures of denaturation, oxidation, aggregation oraggregation, stability of activity can be measured by the binding orfunctional assays disclosed herein, methods of measuring protein serumhalf life are known.

Preferably the p75NTR(NBP) connected to one or more auxiliary moleculescan be expressed at high levels from variety of mammalian host cells toprovide a single species and can be efficiently purified by affinitychromatography for example by binding to Staphylococcus aureus proteinA. Preferably the p75NTR(NBP) connected to one or more auxiliarymolecules can dimerise and preferably the dimer has increased affinityto neurotrophins NGF, BDNF, NT3 or NT4/5 in comparison to p75NTR(NBP)not so connected. Tighter binding has the advantage of higher potencyand a higher therapeutic efficacy as judged by the p75NTR(NBP) effectsfor example as determined by neurotrophin functional assays disclosedherein. Higher potency has the benefit that the p75NTR(NBP) can be usedat lower dosage amounts to achieve the same therapeutic efficacy hencereducing potential toxicity or side effects in-vivo.

Preferably the p75NTR(NBP) of the invention has a half life in-vivo ofabout or more than any one of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 62, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208 or210 hours+/−1 hour, further preferably the p75NTR(NBP) of the inventionhas a half life in-vivo of about or more than 24 hours.

Further preferably the p75NTR(NBP) of the invention has a half lifein-vitro of about or more than any one of 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 62, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208 or 210 days+/−1 day, further preferably the p75NTR(NBP) ofthe invention has a half life in-vitro of about or more than 6 days.Preferably the stability is measured at about physiological pH, in abuffered aqueous solution, preferably at 20° C. or 37° C.

According to the foregoing preferred embodiments, preferably the in-vivohalf life is half life in rat or half life in human, more preferably inhuman. Preferably the half life is determined from serum measurements ofthe levels of p75NTR(NBP) of the invention following administrationin-vivo for example by intravenous or subcutaneous injection.

Further according to the second aspect of the invention the linker ispreferably selected from: (a) a covalent bond, (b) non covalent bond,(c) a peptide bond, (d) one amino acid or a plurality of amino acidscomprising a peptide. Preferably the p75NTR(NBP) is connected to morethan one auxiliary molecule, optionally each auxiliary molecule iseither the same or different or a mixture of the same and different.Further preferably the more than one auxiliary molecule comprises amultimer or a plurality of auxiliary molecules linked to p75NTR(NBP) viaa linker, and wherein each molecule may be there same or different or amixture of the same and different.

Preferably the linker comprises or consists of one or a plurality ofamino acids or comprises or consists of a polypeptide sequence of aminoacids, preferably about 1 to about 25 amino acids, preferably any one of1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids further preferably any one ofabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23 or 24 aminoacids, most preferably 13 amino acids.

Preferably the linker comprises or consists of a polypeptide sequence ofamino acids that lacks any stable secondary structure such as alphahelix, beta strand, 310 helix and pi helix, polyproline helix, alphasheet. Preferably the linker region comprises or consists of apolypeptide sequence of amino acids that defines a flexible or dynamicor unstructured polypeptide such as for example a flexible loop, randomcoil or flexible turn, such unstructured polypeptides are often foundconnecting regions of secondary structure in large protein molecules.

Preferably the linker is a polypeptide sequence of amino acids thatcomprises greater than or about 50% glycine and/or alanine and/or serinein p75NTR(NBP), further preferably greater than or about 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 100% glycine and/or alanine and/orserine in p75NTR(NBP). Preferably the linker region comprises orconsists of a polypeptide sequence of amino acids that comprises bothglycine and serine, preferably with a greater proportion of glycine thatserine, preferably the linker region comprises or consists of flexiblelinkers, SEQ ID NO. 17 (GGGGS)n (n=1 to 4), or helical linkers, SEQ IDNO. 18 (EAAAK)n (n=2 to 5) or polypeptide linkers predominantlyincluding amino acid residues selected from glycine, serine, alanine,and threonine ranging from 1 to 10 repeats of each amino acid and anycombination thereof.

Preferably the linker overcomes or prevents steric hindrance from theauxiliary molecule which could interfere with the aforementionedneurotrophin binding ability or biological activity of the p75NTR(NBP)when compared to p75NTR(NBP) which is not linked to an auxiliarymolecule. Hence the linker region preferably permits flexibility betweenthe p75NTR(NBP) and the auxiliary molecule and allows retention of orimprovement of the aforementioned biological activity of p75NTR(NBP) incomparison to free or native p75NTR(NBP) not so linked as determined bybinding to neurotrophins using binding assays such as described herein.

Further preferably the linker is immunologically inert, such that itdoes not trigger complement mediated lysis, does not stimulateantibody-dependent cell mediated cytotoxicity (ADCC), does not activatemicroglia or T-cells. Preferably the linker region is reduced in one ormore of these activities.

Further preferably the linker comprises or consists of a polypeptideknown or predicted from structural analysis or structural prediction tobe a flexible or dynamic or unstructured polypeptide or to lack a stablesecondary structure.

Most preferably the linker comprises or consists of a polypeptide ofsequence of SEQ ID NO. 19 (GGGGS).

The p75NTR(NBP) of the invention may also comprise a proteolyticcleavage site, optionally interposed between the p75NTR(NBP) and theauxiliary molecule. The proteolytic cleavage site may be located in thelinker or at the junction of the linker with either the p75NTR(NBP)or/and the auxiliary molecule. The p75NTR(NBP) may optionally be cleavedfrom the auxiliary molecule prior to formulation and or administrationfor therapeutic purposes.

Preferably the linker and/or the one or more auxiliary molecules do notimpair or significantly impair the p75NTR(NBP):

(a) effect on the functional activity of the neurotrophins (defined asmodulating or up or down regulating the functional activity of theneurotrophins) NGF, BDNF, NT3 or NT4/5,

(b) binding affinity for any of NGF, BDNF, NT3 or NT4/5 with a bindingaffinity of between about 0.1 nM to about 50 nM

(c) ability to binds to each of the neurotrophins NGF, NT3, BDNF andNT4/5, preferably human NGF, NT3, BDNF and NT4/5.

According to a preferred embodiment of aspects one and two of thepresent invention there is provided a p75NTR(NBP) of the presentinvention there is provided a p75NTR(NBP) for use in treating painwherein the p75NTR(NBP) consists of: (A) a P75NTR(NBP) of sequence SEQID NO. 3, and optionally (B) an immunoglobulin Fc, and furtheroptionally consisting of sequence SEQ ID NO. 11 and optionally (C) alinker, optionally consisting of SEQ ID NO. 19.

According to a third aspect of the invention there is provided a nucleicacid molecule encoding the p75NTR(NBP) or p75NTR(NBP) connected to oneor more auxiliary molecules according to the first or second aspects.Preferably the nucleic acid molecule is for use in the treatment ofpain.

According to a preferred embodiment of the present invention the nucleicacid molecule may further comprise a region encoding a signal sequence,preferably a p75NTR signal sequence for example a DNA or RNA sequence.

According to a fourth aspect of the invention there is provided areplicable expression vector for transfecting a cell, the vectorcomprising the nucleic acid molecule of the third aspect, preferably thevector is a viral vector. Preferably the vector is for use in thetreatment of pain.

Further according to the third or fourth aspects of the invention thereis provided a method of expressing the nucleic acid molecule or thevector of the invention to produce or secrete the p75NTR(NBP) orp75NTR(NBP) connected to one or more auxiliary molecules. Preferably themethod comprises the introduction of the nucleic acid molecule or vectorinto a cell and expression of the nucleic acid therein to produce orsecrete the p75NTR(NBP) or p75NTR(NBP) connected to one or moreauxiliary molecules. Preferably the nucleic acid molecule or vector isintroduced into the cell in-vitro alternatively in-vivo. Preferably theexpressed p75NTR(NBP) or p75NTR(NBP) connected to one or more auxiliarymolecules is expressed in-vitro, optionally further isolated andpurified, alternatively preferably the expressed p75NTR(NBP) orp75NTR(NBP) connected to one or more auxiliary molecules is expressedin-vivo, preferably the in-vivo expression constitutes gene therapy.Preferably the vector is a replicable expression vector, optionally fortransfecting a mammalian cell, preferably the vector is a viral vector.

According to a fifth aspect of the invention there is provided a hostcell harbouring the nucleic acid molecule or vector of either the thirdor fourth aspect, preferably the cell is a mammalian cell.

According to a sixth aspect of the invention there is provided thep75NTR(NBP) for use according to the first or second aspect or thepreferred embodiments thereof, or the nucleic acid or vector for useaccording to the third and fourth aspects wherein, the pain or symptomof pain is selected from:

(a) acute pain and/or spontaneous pain,

(b) chronic pain and or on-going pain,

(c) inflammatory pain including any one of arthritic pain, painresulting from osteoarthritis or rheumatoid arthritis, resulting frominflammatory bowel diseases, psoriasis and eczema

(d) nociceptive pain,

(e) neuropathic pain, including painful diabetic neuropathy or painassociated with post-herpetic neuralgia,

(f) hyperalgesia,

(g) allodynia,

(h) central pain, central post-stroke pain, pain resulting from multiplesclerosis, pain resulting from spinal cord injury, or pain resultingfrom Parkinson's disease or epilepsy,

(i) cancer pain,

(j) post-operative pain,

(k) visceral pain, including digestive visceral pain and non-digestivevisceral pain, pain due to gastrointestinal (GI) disorders, painresulting from functional bowel disorders (FBD), pain resulting frominflammatory bowel diseases (IBD), pain resulting from dysmenorrhea,pelvic pain, cystitis, interstitial cystitis or pancreatitis,

(l) musculo-skeletal pain, myalgia, fibromyalgia, spondylitis,sero-negative (non-rheumatoid) arthropathies, non-articular rheumatism,dystrophinopathy, Glycogenolysis, polymyositis, pyomyositis,

(m) heart or vascular pain, pain due to angina, myocardical infarction,mitral stenosis, pericarditis, Raynaud's phenomenon, scleredoma,scleredoma or skeletal muscle ischemia,

(n) head pain including migraine, migraine with aura, migraine withoutaura cluster headache, tension-type headache.

(o) orofacial pain, including dental pain, temporomandibular myofascialpain or tinnitus, or

(p) back pain, bursitis, menstrual pain, migraine, referred pain,trigeminal neuralgia, hypersensitisation, pain resulting from spinaltrauma and/or degeneration or stroke.

According to a seventh aspect of the invention there is provided thep75NTR(NBP) for use according to the first or second aspect or thepreferred embodiments thereof, or the nucleic acid molecule or vectorfor use according to the third and fourth aspects wherein thep75NTR(NBP) or the nucleic acid molecule or vector is for separate,sequential or simultaneous use in a combination combined with a secondpharmacologically active compound. Preferably the secondpharmacologically active compound of the combination is selected from;

-   -   an opioid analgesic, e.g. morphine, heroin, hydromorphone,        oxymorphone, levorphanol, levallorphan, methadone, meperidine,        fentanyl, cocaine, codeine, dihydrocodeine, oxycodone,        hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone,        naltrexone, buprenorphine, butorphanol, nalbuphine or        pentazocine;    -   a nonsteroidal antiinflammatory drug (NSAID), e.g. aspirin,        diclofenac, diflusinal, etodolac, fenbufen, fenoprofen,        flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen,        ketorolac, meclofenamic acid, mefenamic acid, meloxicam,        nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine,        oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac,        tolmetin or zomepirac;    -   a barbiturate sedative, e.g. amobarbital, aprobarbital,        butabarbital, butabital, mephobarbital, metharbital,        methohexital, pentobarbital, phenobartital, secobarbital,        talbutal, theamylal or thiopental;    -   a benzodiazepine having a sedative action, e.g.        chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam,        oxazepam, temazepam or triazolam;    -   an H₁ antagonist having a sedative action, e.g. diphenhydramine,        pyrilamine, promethazine, chlorpheniramine or chlorocyclizine;    -   a sedative such as glutethimide, meprobamate, methaqualone or        dichloralphenazone;    -   a skeletal muscle relaxant, e.g. baclofen, carisoprodol,        chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenadine;    -   an NMDA receptor antagonist, e.g. dextromethorphan        ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan        ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine,        pyrroloquinoline quinine,        cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid, budipine,        EN-3231 (MorphiDex®, a combination formulation of morphine and        dextromethorphan), topiramate, neramexane or perzinfotel        including an NR2B antagonist, e.g. ifenprodil, traxoprodil or        (−)-(R)-6-{2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl-3,4-dihydro-2(1H)-quinolinone;    -   an alpha-adrenergic, e.g. doxazosin, tamsulosin, clonidine,        guanfacine, dexmetatomidine, modafinil, or        4-amino-6,7-dimethoxy-2-(5-methane-sulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl)        quinazoline;    -   a tricyclic antidepressant, e.g. desipramine, imipramine,        amitriptyline or nortriptyline;    -   an anticonvulsant, e.g. carbamazepine, lamotrigine, topiratmate        or valproate;    -   a tachykinin (NK) antagonist, particularly an NK-3, NK-2 or NK-1        antagonist, e.g.        (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]-naphthyridine-6-13-dione        (TAK-637),        5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one        (MK-869), aprepitant, lanepitant, dapitant or        3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine        (2S,3S);    -   a muscarinic antagonist, e.g oxybutynin, tolterodine,        propiverine, tropsium chloride, darifenacin, solifenacin,        temiverine and ipratropium;    -   a COX-2 selective inhibitor, e.g. celecoxib, rofecoxib,        parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib;    -   a coal-tar analgesic, in particular paracetamol;    -   a neuroleptic such as droperidol, chlorpromazine, haloperidol,        perphenazine, thioridazine, mesoridazine, trifluoperazine,        fluphenazine, clozapine, olanzapine, risperidone, ziprasidone,        quetiapine, sertindole, aripiprazole, sonepiprazole,        blonanserin, iloperidone, perospirone, raclopride, zotepine,        bifeprunox, asenapine, lurasidone, amisulpride, balaperidone,        palindore, eplivanserin, osanetant, rimonabant, meclinertant,        Miraxion® or sarizotan;    -   a vanilloid receptor agonist (e.g. resinferatoxin) or antagonist        (e.g. capsazepine);    -   a beta-adrenergic such as propranolol;    -   a local anaesthetic such as mexiletine;    -   a corticosteroid such as dexamethasone;    -   a 5-HT receptor agonist or antagonist, particularly a        5-HT_(1B/1D) agonist such as eletriptan, sumatriptan,        naratriptan, zolmitriptan or rizatriptan;    -   a 5-HT_(2A) receptor antagonist such as        R(+)-alpha-(2,3-dimethoxy-phenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol        (MDL-100907);    -   a cholinergic (nicotinic) analgesic, such as ispronicline        (TC-1734), (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine        (RJR-2403), (R)-5-(2-azetidinylmethoxy)-2-chloropyridine        (ABT-594) or nicotine;    -   Tramadol®;    -   a PDEV inhibitor, such as        5-[2-ethoxy-5-(4-methyl-1-piperazinyl-sulphonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one        (sildenafil),        (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′,1′:6,1]-pyrido[3,4-b]indole-1,4-dione        (IC-351 or tadalafil),        2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one        (vardenafil),        5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-c]pyrimidin-7-one,        5-(5-acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,        5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,        4-[(3-chloro-4-methoxybenzyl)amino]-2-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide,        3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide;    -   a cannabinoid;    -   metabotropic glutamate subtype 1 receptor (mGluR1) antagonist;    -   a serotonin reuptake inhibitor such as sertraline, sertraline        metabolite demethylsertraline, fluoxetine, norfluoxetine        (fluoxetine desmethyl metabolite), fluvoxamine, paroxetine,        citalopram, citalopram metabolite desmethylcitalopram,        escitalopram, d,l-fenfluramine, femoxetine, ifoxetine,        cyanodothiepin, litoxetine, dapoxetine, nefazodone, cericlamine        and trazodone;    -   a noradrenaline (norepinephrine) reuptake inhibitor, such as        maprotiline, lofepramine, mirtazepine, oxaprotiline, fezolamine,        tomoxetine, mianserin, buproprion, buproprion metabolite        hydroxybuproprion, nomifensine and viloxazine (Vivalan®),        especially a selective noradrenaline reuptake inhibitor such as        reboxetine, in particular (S,S)-reboxetine;    -   a dual serotonin-noradrenaline reuptake inhibitor, such as        venlafaxine, venlafaxine metabolite O-desmethylvenlafaxine,        clomipramine, clomipramine metabolite desmethylclomipramine,        duloxetine, milnacipran and imipramine;    -   an inducible nitric oxide synthase (iNOS) inhibitor such as        S-[2-[(1-iminoethyl)amino]ethyl]-L-homocysteine,        5-[2-[(1-iminoethyl)-amino]ethyl]-4,4-dioxo-L-cysteine,        S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine,        (2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic        acid,        2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)-butyl]thio]-5-chloro-3-pyridinecarbonitrile;        2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-4-chlorobenzonitrile,        (2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiazolebutanol,        2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-6-(trifluoromethyl)-3        pyridinecarbonitrile,        2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonitrile,        N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-carboxamidine,        or guanidinoethyldisulfide;    -   an acetylcholinesterase inhibitor such as donepezil;    -   a prostaglandin E2 subtype 4 (EP4) antagonist such as        N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl}amino)-carbonyl]-4-methylbenzenesulfonamide        or        4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]benzoic        acid;    -   a leukotriene B4 antagonist; such as        1-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylic        acid (CP-105696),        5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E-hexenyl]oxyphenoxy]-valeric        acid (ONO-4057) or DPC-11870,    -   a 5-lipoxygenase inhibitor, such as zileuton,        6-[(3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl])phenoxy-methyl]-1-methyl-2-quinolone        (ZD-2138), or 2,3,5-trimethyl-6-(3-pyridylmethyl),        1,4-benzoquinone (CV-6504);    -   a sodium channel blocker, such as lidocaine; or    -   a 5-HT3 antagonist, such as ondansetron;        and the pharmaceutically acceptable salts and solvates thereof.

According to an eighth aspect of the present invention there is provideda method of treating, preventing, ameliorating, controlling, reducingincidence of, or delaying the development or progression of pain or anyof the foregoing pain and/or symptoms of pain in an individual,comprising administration to the individual of an effective amount ofthe p75NTR(NBP) according to the first, second or seventh aspect or thepreferred embodiments thereof, or the nucleic acid molecule or vectoraccording to the third and fourth aspects.

Preferably the individual is a mammal, for example a companion animalsuch as a horse, cat or dog or a farm animal such as a sheep, cow orpig. Most preferably the mammal is a human.

According to a ninth aspect of the present invention there is provided apharmaceutical composition for any one or more of treating, preventing,ameliorating, controlling, reducing incidence of, or delaying thedevelopment or progression of pain or any of the foregoing pain/orsymptoms, comprising the p75NTR(NBP) according to the first, second orseventh aspect or the preferred embodiments thereof, or the nucleic acidmolecule or vector according to the third and fourth aspects and apharmaceutically acceptable carrier and/or an excipient.

Preferably the p75NTR(NBP) according to the first, second or seventhaspects or the preferred embodiments thereof, or the nucleic acidmolecule or vector according to the third and fourth aspects or thepharmaceutical composition of the ninth aspect is prepared for orsuitable for oral, sublingual, buccal, topical, rectal, inhalation,transdermal, subcutaneous, intravenous, intra-arterial, intramuscular,intracardiac, intraosseous, intradermal, intraperitoneal, transmucosal,vaginal, intravitreal, intra-articular, peri-articular, local orepicutaneous administration.

Preferably the p75NTR(NBP) according to the first, second or seventhaspect or the preferred embodiments thereof, or the nucleic acidmolecule or vector according to the third and fourth aspects or thepharmaceutical composition of the ninth aspect is prepared for orsuitable for administration prior to and/or during and/or after theonset of pain or for such use.

Preferably the p75NTR(NBP) according to the first, second or seventhaspect or the preferred embodiments thereof, or the nucleic acidmolecule or vector according to the third and fourth aspects or thepharmaceutical composition of the ninth aspect is for or prepared foradministration between once to 7 times per week, further preferablybetween once to four times per month, further preferably between once tosix times per 6 month period, further preferably once to twelve timesper year. Preferably the medicament is to be or prepared to beperipherally administered in a period selected from: once daily, onceevery two, three, four, five or six days, weekly, once every two weeks,once every three weeks, monthly, once every two months, once every threemonths, once every four months, once every five months, once every sixmonths, once every seven months, once every eight months, once everynine months, once every ten months, once every eleven months or yearly.

Further preferably the p75NTR(NBP) according to the first, second orseventh aspect or the preferred embodiments thereof, or the nucleic acidmolecule or vector according to the third and fourth aspects or thepharmaceutical composition of the ninth aspect is to be or prepared tobe peripherally administered via a route selected from one or more of;orally, sublingually, buccally, topically, rectally, via inhalation,transdermally, subcutaneously, intravenously, intra-arterially orintramuscularly, via intracardiac administration, intraosseously,intradermally, intraperitoneally, transmucosally, vaginally,intravitreally, epicutaneously, intra-articularly, peri-articularly orlocally.

Preferably the p75NTR(NBP) according to the first, second or seventhaspect or the preferred embodiments thereof, or the nucleic acidmolecule or vector according to the third and fourth aspects or thepharmaceutical composition of the ninth aspect is for or is prepared foradministration at a concentration of between about 0.1 to about 200mg/ml; preferably at any one of about 0.5, 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190 or 200 mg/ml+/−about 10% error, most preferablyat about 50 mg/ml.

Preferably the p75NTR(NBP) according to the first, second or seventhaspect or the preferred embodiments thereof, or the nucleic acidmolecule or vector according to the third and fourth aspects or thepharmaceutical composition of the ninth aspect is for or is prepared foradministration at a concentration of between about 0.1 to about 200mg/kg of body weight; preferably at any one of about 0.5, 1, 5, 10, 1520, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190 or about 200 mg/kg of bodyweight+/−about 10% error, most preferably at about 10 mg/kg.

According to a tenth aspect of the present invention there is provided akit comprising:

(a) the p75NTR(NBP) according to the first, second or seventh aspect orthe preferred embodiments thereof, or the nucleic acid molecule orvector according to the third and fourth aspects or the pharmaceuticalcomposition of the ninth aspect; and

(b) instructions for the administration of an effective amount of saidp75NTR(NBP), nucleic acid molecule, vector or pharmaceutical compositionto an individual for any one or more of the prevention or treatment ofpain and/or symptoms of pain or for ameliorating, controlling, reducingincidence of, or delaying the development or progression of pain and/orsymptoms of pain.

The kit may include one or more containers containing the p75NTR(NBP),nucleic acid, vector or pharmaceutical composition described herein andinstructions for use in accordance with any of the methods and uses ofthe invention. The kit may further comprise a description of selectingan individual suitable for treatment based on identifying whether thatindividual has a pain or a symptom of pain or is at risk of having such.The instructions for the administration of the pharmaceuticalcomposition may include information as to dosage, dosing schedule androutes of administration for the intended treatment.

According to an eleventh aspect of the present invention there isprovided the p75NTR(NBP) according to the first, second or seventhaspect or the preferred embodiments thereof, or the nucleic acidmolecule or vector according to the third and fourth aspects or thepharmaceutical composition of the ninth aspect for use in any one ormore of the prevention or treatment or for ameliorating, controlling,reducing incidence of, or delaying the development or progression of acondition or the symptoms of a condition associated with any one or moreof the neurotrophins NGF, BDNF, NT-3, NT-4/5.

NGF (Nerve growth factor) binds with at least two classes of receptors:the p75NTR and TrkA, a transmembrane tyrosine kinase, it is involved inaxonal growth, branching and elongation. Conditions and symptomsassociated with NGF are known. NGF is expressed in and associated withinflammatory conditions and pain [Protein Sequence NP_002497.2,NP_038637]. Also, NGF has been shown to play a role in numbercardiovascular diseases, such as coronary atherosclerosis, obesity, type2 diabetes, and metabolic syndrome as well as in Multiple Sclerosis.Reduced plasma levels of NGF (and also of BDNF) have been associatedwith acute coronary syndromes and metabolic syndromes. NGF is alsorelated to various psychiatric disorders, such as dementia, depression,schizophrenia, autism, Rett syndrome, anorexia nervosa, and bulimianervosa and has also been implicated in development of Alzheimer'sdisease and neurodegenerative disorders. NGF has also been shown toaccelerate wound healing and there is evidence that it could be usefulin the treatment of skin ulcers and corneal ulcers, it has been shown toreduce neural degeneration and to promote peripheral nerve regenerationin rats.

BDNF (brain-derived neurotrophic factor) is a neurotrophin whichsupports neuronal survival and growth during development of the nervoussystem [Protein Sequence NP_001137277.1, NP_001041604]. BDNF binds cellsurface receptors TrkB and p75NTR and also modulates the activity ofAlpha-7 nicotinic receptor. Conditions and symptoms associated with BDNFare known. BDNF has been shown to play a significant role in thetransmission of physiologic and pathologic pain, particularly in modelsof acute pain, inflammatory pain and neuropathic pain, where BDNFsynthesis is found to be greatly increased; also BDNF has been shown tobe up-regulated in conditions of chronic pain as well as furtherconditions such as eczema and psoriasis. Down-regulation of BDNF is seenin depression, schizophrenia, obsessive-compulsive disorder, Alzheimer'sdisease, Huntington's disease, Rett syndrome, and dementia, as well asanorexia nervosa and bulimia nervosa.

Neurotrophin-4 (NT-4), also known as neurotrophin-5 (NT-5), is aneurotrophic factor that signals predominantly through the p75NTR andTrkB receptors and promotes the survival of peripheral sensorysympathetic neurons. The mature peptide of this protein is identical inall mammals examined including human, pig, rat and mouse. [ProteinSequence NP_006170, NP_937833]. NT-4 is synthesized by most neurons ofthe dorsal root ganglion (DRG) and those in the paravertebral andprevertebral sympathetic ganglia, spinal dorsal and ventral horn and isfound expressed in many tissues including the prostate, thymus, placentaand skeletal muscle. Conditions and symptoms associated with NT-4/5 areknown. Defects in NT4/5 are associated with susceptibility to primaryopen angle glaucoma. Neurotrophin 4 has also been shown to contribute tobreast cancer cell survival and is a target to inhibit tumour growth.NT-4/5 is known to be involved in pain-signalling systems such asnociceptive pain, upregulation of NT-4/5 is also seen in chronicinflammatory conditions of the skin, such as dermatitis, eczema, prurigolesions of atopic dermatitis. Down regulation of NT-4/5 is seen inAlzheimer's Disease, Huntington's disease.

Neurotrophin-3 (NT-3), is a neurotrophin that is structurally related tobeta-NGF, BDNF, and NT-4, and that controls survival and differentiationof mammalian neurons and the maintenance of the adult nervous system,and may affect development of neurons in the embryo when it is expressedin human placenta. Conditions and symptoms associated with NT3 areknown. NTF3-deficient mice generated by gene targeting display severemovement defects of the limbs. NT-3 signals through the Trk receptorsand promotes the growth and survival of nerve and glial cells [ProteinSequence NP_001096124.1 and NP_032768]. The amino acid sequences ofhuman, Mouse and rat NT-3 are identical. NT3 and its cognate receptor,tyrosine kinase C (TrkC), are known to modulate neuropathic pain andnociceptive pain and the mechanism of nociception and proporioception,for example NT3 expression is increased in the small DRG cells ofneuropathic animals. NT3 expression is also associated with neuropathiessuch as diabetic polyneuropathy and HIV-related neuropathy, large fiberneuropathy including atrophy, it is further involved in the developmentof hyperalgesia (a decrease in the threshold of a normally noxiousstimuli), allodynia (a non-noxious stimulus becomes noxious), andspontaneous pain (pain in the apparent absence stimuli) and is a knownmodulator of muscle pain.

The invention will now be described by reference to the followingexamples which are provided to illustrate, but not to limit, theinvention.

The following Examples are provide to illustrate not to limit theinvention.

EXAMPLES Example 1. Endogenous p75NTR(NBP)-Neurotrophic Complex isDetected to Human Plasma

An assay utilizing magnetic bead based NGF (Millipore Corp; MA) andimmunoprecipitation using biotinylated goat polyclonal anti-human NGFantibody (Novus Biologicals; CO) followed by SISCAPA (Siscapahttp://siscapa.com/home.html) then subsequently determined in LC-MS/MS(LLOQ 7 pg/ml) was employed to provide direct evidence for the existenceof a p75NTR/NGF complex in human plasma. The polyclonal anti-NGFantibody, was used to quantify free NGF in human serum/plasma.

Peptide sequences (CAYGYYQDETTGR (SEQ ID NO. 4) VCEAGSGLVFSCQDK (SEQ IDNO. 6) WADAECEEIPGR (SEQ ID NO. 7)) of the extracellular domain ofp75NTR (as shown in FIG. 2) were selected as p75NTR standards byLC-MS/MS following chimera digestion of p75NTR. Co-immunoprecipitationof p75NTR detected in the total NGF assay (as described above) was ableto demonstrate the presence of the p75NTR control peptides providingevidence of a soluble p75-NGF complex in human serum. Similarinteractions of p75NTR(NBP) and BDNF, NT-3 and NT4/5 were alsodetermined using co-immunoprecipitation with respective BDNF, NT-3 andNT4/5 antibodies. These studies demonstrate the presence of a solublep75 neurotrophic binding protein in the plasma of humans bound to eitherNGF, BDNF, NT-3 and NT4/5.

The data presented in FIG. 2 shows soluble p75NTR(NBP) peptide standardfragments and co-immunoprecipitation of p75NTR(NBP) and NGFdemonstrating p75NTR(NBP)-NGF complex in human plasma. It is evidentfrom the findings of p75NTR(NBP)-NGF co-immunoprecipitation studies inhuman plasma, that the key peptides involved in binding in theneurotrophin-p75NTR(NBP) complex are: WADAECEEIPGR (SEQ ID NO. 7),CAYGYYQDETTGR (SEQ ID NO. 4), VCEAGSGLVFSCQDK (SEQ ID NO. 6),additionally we have determined that the sequence LDSVTSDVVSATEPCKP (SEQID NO. 8) is also important in binding.

The experiments were repeated with BDNF and NT-3 showing the importanceof the same key binding regions of p75NTR(NBP) and demonstrating theco-immunoprecipitation of p75NTR(NBP) with BDNF and NT-3 demonstratingp75NTR(NBP)-BDNF and p75NTR(NBP)-NT-3 complexes in human plasma.

Example 2. Soluble p75NTR(NBP) Inhibits NGF Function in U20S Cell LineExpressing TrkA and in U20S Cell Line Co-Expressing TrkA and p75NTR

The effects of p75NTR(NBP) on NGF function was explored in U20S cellsexpressing TrkA and U20S cells co-expressing TrkA and p75NTR (DiscoverxCorp CA). The assay was run in accordance with the PathHuntermethodology (Discoverx Corp CA) briefly, U20S cells expressing TrkA andTrkA+p75NTR were purchased from DiscoveRx Corporation. The U20S cellsexpressing TrkA and cells co-expressing TrkA+p75NTR were plated inminimum essential medium (MEM; Sigma Aldrich)+0.5% horse serum (SigmaAldrich) at a density of 40,000 cells/well and incubate at 37° C. overnight. The plates were removed from the incubator 30 min prior to useand stored at room temperature.

NGFα (Sigma Aldrich) and p75NTR(NBP)-Fc (p75NTR(NBP) SEQ ID NO. 2 coupleto IgG-Fc SEQ ID NO. 12 using linker SEQ ID NO 17) or p75NTR(NBP)(p75NTR(NBP) SEQ ID NO. 2) or p75NTR(NBP) coupled to albumin, SEQ ID NO.10, or p75NTR(NBP) (p75NTR(NBP) SEQ ID NO. 2) coupled to transferrin,SEQ ID NO. 9, were diluted in Hanks Balanced Salt Solution Buffer (HBSSSigma Aldrich) plus 0.25% BSA. NGF was serially diluted to giveconcentrations ranging from 0 nM to 200 nM. P75NTR(NBP) coupled toeither Fc IgG, albumin or transferrin was diluted in HBSS to give aconcentration of p75NTR of 100 ng/ml.

5 μl of each of the NGF concentrations (0-200 nM) and 5 μl ofp75NTR(NBP)+/−carrier were added to each well. In an attempt tounderstand the impact on pre-incubation p75NTR(NBP)+/−carrier and NGFwere allowed to incubate at room temperature for 20 minutes prior toadding 10 μl of this mixture to each well. Immediately following theaddition of NGF and p75NTR(NBP)+/−carrier 12 μl of detection solution(Discoverx Corp CA) was added to each well and the plate was allowed toincubate at room temperature for 60 minutes prior to reading on aParkard Victor 2 plate reader. Dose response curves and shifts in thecurves in response to the addition of p75NTR(NBP) were observed.Regardless of the carrier used p75NTR(NBP) without a carrier,p75NTR(NBP)-Fc, p75NTR(NBP)-albumin or p75NTR(NBP)-transferrinsuppression and shifts in the NGF dose response curves were observed(see FIG. 4).

Example 3. p75NTR(NBP) Inhibits BDNF Function in U20S Cell LineExpressing TrkB and in U20S Cell Line Co-Expressing TrkB and p75NTR

The effects of p75NTR(NBP) on BDNF function was explored in U20S cellsexpressing TrkB and U20S cells co-expressing TrkB and p75NTR (DiscoverxCorp CA). The assay was run in accordance with the PathHuntermethodology (Discoverx Corp CA) briefly, U20S cells expressing TrkB andTrkB+p75NTR were purchased from DiscoveRx Corporation. The U20S cellsexpressing TrkB and cells co-expressing TrkB+p75NTR were plated inminimum essential medium (MEM; Sigma Aldrich)+0.5% horse serum (SigmaAldrich) at a density of 40,000 cells/well and incubate at 37° C.overnight. The plate were removed from the incubator 30 minutes prior touse and stored at room temperature.

BDNFα (Sigma Aldrich) and p75NTR(NBP)-Fc (p75NTR(NBP) SEQ ID NO. 2couple to IgG-Fc SEQ ID NO. 12 using linker SEQ ID NO 17) or p75NTR(NBP)(p75NTR(NBP) SEQ ID NO. 2) or p75NTR(NBP) coupled to albumin, SEQ ID NO.10, or p75NTR(NBP) (p75NTR(NBP) SEQ ID NO. 2) coupled to transferrin,SEQ ID NO. 9, were diluted in Hanks Balanced Salt Solution Buffer (HBSSSigma Aldrich) plus 0.25% BSA. BDNF was serially diluted to giveconcentrations ranging from 0 nM to 200 nM. P75NTR(NBP) coupled toeither Fc-IgG, albumin or transferrin was diluted in HBSS to give aconcentration of p75NTR(NBP) 100 ng/ml.

5 μl of each of the BDNF concentrations (0-200 nM) and 5 μl ofp75NTR(NBP)+/−carrier were added to each well. In an attempt tounderstand the impact on pre-incubation p75NTR(NBP)+/−carrier and BDNFwere allowed to incubate at room temperature for 20 minutes prior toadding 10 μl of this mixture to each well Immediately following theaddition of BDNF and p75NTR(NBP)+/−carrier 12 μl of detection solution(Discoverx Corp CA) was added to each well and the plate was allowed toincubate at room temperature for 60 minutes prior to reading on aParkard Victor 2 plate reader. Dose response curves and shifts in thecurves in response to the addition of p75NTR(NBP) were observed.Regardless of the carrier used p75NTR(NBP) without a carrier,p75NTR(NBP)-Fc, p75NTR(NBP)-albumin or p75NTR(NBP)-transferrinsuppression and shifts in the BDNF dose response curves were observed(see FIG. 5).

Example 4

Biocore data demonstrating p75NTR(NBP) binding to BDNF and competitionbetween anti-BDNF and p75NTR(NBP). P75-Fc (p75NTR(NBP)-Fc), (500 μg/ml)was diluted to a concentration of 25 μg/ml in 10 mM acetate pH 5.0 andimmobilised on the Biocore chip utilising the running buffer HBS-P(0.005% P20)+1 mg/ml BSA. A stock solution of BDNF (from R and Dsystems) 2 uM in HBS-P (0.005% P20)+1 mg/ml BSA buffer was prepared.BDNF competition experiment was preformed with p75 and presence orabsence of MAB248 (BDNF anti-body RandD systems) at the followingconcentrations: i) MAB248 (500 nM) alone, ii) BDNF (20 nM)+MAB248 (500nM) iii) BDNF (20 nM) alone and iv) BDNF (20 nM)+MAB248 (100 nM). Allsamples were prepared from stock solutions and diluted in runningbuffer; HBS-P (0.005% P20)+1 mg/ml BSA. All samples were left at roomtemperature for 30 minutes prior to injection. A manual sensorgram wasinitiated across the biocore chip at a flow rate of 50 ul/min. Eachsample was injected for 30 seconds in the order listed above in aseparate sensorgram cycle. Regeneration was carried out where necessarywith 5 mM NaOH for 12 seconds (10 ul) cycle. As depicted in FIG. 6p75NTR(NBP) binds BDNF.

Example 5. p75NTR(NBP) Inhibition of NGF Activity in PC12 Cells

The p75NTR(NBP) was demonstrated to significantly inhibit the activityof NGF in PC-12 and N2OS cells expressing TrkA and/or TrkA plus p75 onthe cell surface, FIG. 7.

Low-passage PC12 cells (passage number 20-24) were grown directly on96-well plates in Dulbecco's modified Eagle's medium (DMEM) supplementedwith 5% fetal bovine serum and 10% heat-inactivated horse serum andpenicillin-streptomycin (10 U/ml-0.1 mg/ml), and incubated in ahumidified atmosphere containing 5% CO2, 37° C. NGF (50 ng/ml) was addedto the growth medium to differentiate the PC12 cells. A doseconcentration of P75NTR(NBP) of 200, 100, 50, 25, 12.5, 6.25, 3.1, 1.5,0.78, 0.39, 0.2, 0.1 ng/ml was added to each well across the plate. Theplate was incubated at 37° C. for 24 hours following which the cellnumbers were determined by a cell counter. As shown in FIG. 8 theaddition of p75NTR(NBP) to the media blocked significantly thedifferentiation of PC-12 cells to their axonal phenotype. In thepresence of NGF PC-12 cells differentiate and form a neuronal likephenotype. This was inhibited in a dose dependent manner in response top75NTR(NBP). It was concluded from these studies that p75NTR(NBP) blocksthe action of NGF.

Example 6. Neurotrophic Binding Protein p75NTR(NBP) Inhibits BDNF Effectin Mechanical Hyperalgesia in a Model of Nerve Excitability

Male Sprague Dawley rats were anaesthetised with isoflurane (5% inoxygen for induction, 1.5-2.0% in oxygen for maintenance). The spinalcord was exposed by lumbar laminectomy to uncover the L4-L6 region ofthe cord, and a tungsten microelectrode inserted into the dorsal horn torecord from an isolated wide dynamic range neurone. Individual WDRneurones were isolated such that the spike amplitude of the recordedunit was at least 3× the level of the baseline noise. Responses wereamplified using a neurolog AC-coupled amplifier and recorded using CEDspike2 software. Prior to drug application, 3 stable baseline responses(<10% variation) were obtained to a range of natural stimuli (brush, vonFrey, pinch and heat). Brush stimulus was applied by stroking the centreof the peripheral receptive field with a fine cotton wisp. Von Freyfilament and heat (using a constant water jet set at 43 degrees) wereapplied for a period of 10 seconds. Pinch was applied for a period of 5seconds. Once stable baseline responses were obtained, BDNF alone (500ng in 0.1% BSA) or BDNF (500 ng)+p75NTR(NBP) (500 ng pre-incubated for40 minutes prior to injection) was applied to the centre of thereceptive field in a volume of 25 ul using an insulin syringe. Drugeffects were followed for 150 minutes and tests carried out at 10 minintervals. At the termination of the experiment the rat was culled bycervical dislocation.

As demonstrated in FIG. 8 BDNF caused a significant increase in neuronalexcitability and this was amplified following stimulus. The addition ofp75NTR(NBP) significant reduced neuronal excitability in response to allstimulus; saline, BDNF, brush and pinch. It is evident that p75NTR(NBP)reduces neuronal excitability and subsequently pain.

Example 7—Neurotrophic Binding Protein P75NTR(NBP) Inhibits Hyperalgesiain UVIH Model Exposure of Animals to UV

Charles River male Sprague Dawley rats (175-200 g) were anaesthetisedwith a 2% isofluorane O₂ mixture. Anaesthesia was maintained via a nosecone while the plantar surface of the right hind paw was irradiated with300mJ/cm² of UV (Saalmann CupCube system, Saalmann GmbH, Germany;λ=280-400 nm). The source of irradiation was adapted to the plantarsurface of rat's hind paw by using shaped delivery collar (8×12 mm),attached to the UV source (Pfizer Facilities Management and EngineeringTeam). Intensity of UV sources was calibrated using the ABLE 1400Aradiometer with SEL005/WBS320/TD Filter (ABLE Instruments and ControlsLtd). The UV exposure used, causes cutaneous erythema in naïve rats andno other adverse events (no loss in body weight, no sign of stress orobvious discomfort).

Evaluation of Thermal Hyperalgesia

Thermal hyperalgesia was assessed using the rat plantar test (UgoBasile, Italy) following a modified method of Hargreaves et al (1988).Rats were habituated to the apparatus (15-30 min) before testing. Theplantar test consisted of three individual perspex boxes on an elevatedglass table. Two rats were housed in each box so that 6 rats/apparatuscan be tested simultaneously.

A mobile infrared heat source was applied directly below the plantarsurface of the rat hind paw. The paw withdrawal latency (PWL) wasdefined as the time (seconds) taken by the rat to remove its hind pawfrom the heat source. The source was calibrated to give an response of8-12 sec on untreated animals (105-120 mW/cm²). An automatic cut offpoint of 20 sec was set to prevent tissue damage.

Five recordings were taken from each rat hind paws during baselinesmeasurements (naïve rats before UV exposures, Day 0) and confirmation ofhyperalgesia onset (days 1). Two sessions per day were performed on day0 and 1.

Three recordings per paw were instead collected for baselines and eachtesting point during evaluation of efficacy of medicaments on day 2, 3and 4 (48, 72 and 96 hours post UV exposure, respectively). Uninjuredpaw was always assessed first.

Thermal hyperalgesia is defined as animals showing mean PWL≤5 sec, whichare selected for studies.

Evaluation of Static Allodynia

Animals are habituated to wire bottom test cages prior to the assessmentof allodynia. Animals are familiarised with the von Frey Hairs byapplication of 15 g, 8 g+4 g filament on 1^(st) day of habituation, 4 gfilament 2^(nd) day before a full up down measurement is performed.Static allodynia is evaluated by application of von Frey hairs(Stoelting, Wood Dale, Ill., U.S.A.) in ascending order of force (0.6,1, 1.4, 2, 4, 6, 8, 10, 15 and 26 grams) to the plantar surface of hindpaws. Each von Frey hair is applied to the paw for a maximum of 6seconds, or until a withdrawal response occurs. Once a withdrawalresponse to a von Frey hair is established, the paw is re-tested,starting with the filament below the one that produced a withdrawal, andsubsequently with the remaining filaments in descending force sequenceuntil no withdrawal occurs. The highest force of 26 g lifts the paw aswell as eliciting a response, thus representing the cut off point. Eachanimal has both hind paws tested in this manner. The lowest amount offorce required to elicit a response is recorded as paw withdrawalthreshold (PWT) in grams. Static allodynia is defined as present ifanimals respond to a stimulus of, or less than, 4 g, which is innocuousin normal rats.

Animals are baselined prior to testing and those displaying allodyniawhich meets the inclusion criteria are randomized based on PWL and aredosed. Test compound is dosed and at predetermined times (dependant onthe pharmacokinetic properties of the test compound), following dosingmeasurements are made at selected times post dosing and all data iscollected for analysis. As demonstrated in FIG. 9 PWL data for thep75NTR(NBP) 10 mg/kg subcutaneously (denoted NBPP-Fc) is shown incomparison to control, ibuprofen 100 mg/kg po and anti-NGF antibody 10mg/kg subcutaneously, data for evaluation of thermal hyperalgesia showedequivalent measures in each case.

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April; 46(1):41-51

1-23. (canceled)
 24. A kit comprising: (a) a p75NTR neurotrophin binding protein (p75NTR(NBP)) connected to one or more auxiliary molecules selected from the group consisting of transferrin or a portion thereof, albumin or a portion thereof, an immunoglobulin Fc or a portion thereof, and a polyethylene glycol polymer chain, wherein the p75NTR(NBP) binds to any of NGF, BDNF, NT3, or NT4/5 with a binding affinity (K_(d)) of between about 0.1 nM to about 50 nM as measured by surface plasmon resonance at 20° C.; and (b) instructions for the administration of an effective amount of the p75NTR(NBP) to an individual for treating pain and/or a symptom of pain in a subject in need thereof.
 25. A nucleic acid molecule encoding the p75NTR(NBP) according to claim 24, optionally further comprising encoding a signal sequence.
 26. A pharmaceutical composition comprising the nucleic acid molecule according to claim
 25. 27. A replicable expression vector for transfecting a cell, the vector comprising the nucleic acid molecule of claim
 25. 28. The replicable expression vector of claim 27 wherein the vector is a viral vector.
 29. A host cell harboring the nucleic acid molecule of claim
 25. 30. A method for treating pain and/or a symptom of pain in a subject in need thereof, comprising administering to said subject the pharmaceutical composition according to claim
 26. 31. The method according to claim 30, wherein the pain or symptom of pain is selected from: (a) acute pain and/or spontaneous pain, (b) chronic pain and or on-going pain, (c) inflammatory pain including any one of arthritic pain, pain resulting from osteoarthritis or rheumatoid arthritis, resulting from inflammatory bowel diseases, (d) nociceptive pain, (e) neuropathic pain, including painful diabetic neuropathy or pain associated with post-herpetic neuralgia, (f) hyperalgesia, (g) allodynia, (h) central pain, central post-stroke pain, pain resulting from multiple sclerosis, pain resulting from spinal cord injury, or pain resulting from Parkinson's disease or epilepsy, (i) cancer pain, (j) post-operative pain, (k) visceral pain, including digestive visceral pain and non-digestive visceral pain, pain due to gastrointestinal (GI) disorders, pain resulting from functional bowel disorders (FBD), pain resulting from inflammatory bowel diseases (IBD), pain resulting from dysmenorrhea, pelvic pain, cystitis, interstitial cystitis or pancreatitis, (l) musculo-skeletal pain, myalgia, fibromyalgia, spondylitis, sero-negative (non-rheumatoid) arthropathies, non-articular rheumatism, dystrophinopathy, Glycogenolysis, polymyositis, pyomyositis, (m) heart or vascular pain, pain due to angina, myocardical infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredoma, scleredoma or skeletal muscle ischemia, (n) head pain including migraine, migraine with aura, migraine without aura cluster headache, tension-type headache. (o) orofacial pain, including dental pain, temporomandibular myofascial pain or tinnitus, or (p) back pain, bursitis, menstrual pain, migraine, referred pain, trigeminal neuralgia, hypersensitisation, pain resulting from spinal trauma and/or degeneration or stroke.
 32. A method of treating pain and/or a symptom of pain in a subject in need thereof, comprising the sequential or co-administration of the pharmaceutical composition according to claim 26, and a second pharmacologically active compound.
 33. The method according to claim 32, wherein the second pharmacologically active compound of the combination is selected from: an opioid analgesic, e.g. morphine, heroin, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine or pentazocine; a nonsteroidal antiinflammatory drug (NSAID), e.g. aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin or zomepirac; a barbiturate sedative, e.g. amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, phenobartital, secobarbital, talbutal, theamylal or thiopental; a benzodiazepine having a sedative action, e.g. chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam or triazolam; an H₁ antagonist having a sedative action, e.g. diphenhydramine, pyrilamine, promethazine, chlorpheniramine or chlorocyclizine; a sedative such as glutethimide, meprobamate, methaqualone or dichloralphenazone; a skeletal muscle relaxant, e.g. baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenadine; an NMDA receptor antagonist, e.g. dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinine, cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid, budipine, EN-3231 (MorphiDex®, a combination formulation of morphine and dextromethorphan), topiramate, neramexane or perzinfotel including an NR2B antagonist, e.g. ifenprodil, traxoprodil or (−)-(R)-6-{2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl-3,4-dihydro-2(1H)-quinolinone; an alpha-adrenergic, e.g. doxazosin, tamsulosin, clonidine, guanfacine, dexmetatomidine, modafinil, or 4-amino-6,7-dimethoxy-2-(5-methane-sulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl) quinazoline; a tricyclic antidepressant, e.g. desipramine, imipramine, amitriptyline or nortriptyline; an anticonvulsant, e.g. carbamazepine, lamotrigine, topiratmate or valproate; a tachykinin (NK) antagonist, particularly an NK-3, NK-2 or NK-1 antagonist, e.g. (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]-naphthyridine-6-13-dione (TAK-637), 5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one (MK-869), aprepitant, lanepitant, dapitant or 3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine (2S,3S); a muscarinic antagonist, e.g oxybutynin, tolterodine, propiverine, tropsium chloride, darifenacin, solifenacin, temiverine and ipratropium; a COX-2 selective inhibitor, e.g. celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib; a coal-tar analgesic, in particular paracetamol; a neuroleptic such as droperidol, chlorpromazine, haloperidol, perphenazine, thioridazine, mesoridazine, trifluoperazine, fluphenazine, clozapine, olanzapine, risperidone, ziprasidone, quetiapine, sertindole, aripiprazole, sonepiprazole, blonanserin, iloperidone, perospirone, raclopride, zotepine, bifeprunox, asenapine, lurasidone, amisulpride, balaperidone, palindore, eplivanserin, osanetant, rimonabant, meclinertant, Miraxion® or sarizotan; a vanilloid receptor agonist (e.g. resinferatoxin) or antagonist (e.g. capsazepine); a beta-adrenergic such as propranolol; a local anaesthetic such as mexiletine; a corticosteroid such as dexamethasone; a 5-HT receptor agonist or antagonist, particularly a 5-HT_(1B/1D) agonist such as eletriptan, sumatriptan, naratriptan, zolmitriptan or rizatriptan; a 5-HT_(2A) receptor antagonist such as R(+)-alpha-(2,3-dimethoxy-phenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (MDL-100907); a cholinergic (nicotinic) analgesic, such as ispronicline (TC-1734), (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403), (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594) or nicotine; Tramadol®; a PDEV inhibitor, such as 5-[2-ethoxy-5-(4-methyl-1-piperazinyl-sulphonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (sildenafil), (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′,1′:6,1]-pyrido[3,4-b]indole-1,4-dione (IC-351 or tadalafil), 2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one (vardenafil), 5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo [4,3-d]pyrimidin-7-one, 5-(5-acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, 4-[(3-chloro-4-methoxybenzyl)amino]-2-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide, 3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide; a cannabinoid; metabotropic glutamate subtype 1 receptor (mGluR1) antagonist; a serotonin reuptake inhibitor such as sertraline, sertraline metabolite demethylsertraline, fluoxetine, norfluoxetine (fluoxetine desmethyl metabolite), fluvoxamine, paroxetine, citalopram, citalopram metabolite desmethylcitalopram, escitalopram, d,l-fenfluramine, femoxetine, ifoxetine, cyanodothiepin, litoxetine, dapoxetine, nefazodone, cericlamine and trazodone; a noradrenaline (norepinephrine) reuptake inhibitor, such as maprotiline, lofepramine, mirtazepine, oxaprotiline, fezolamine, tomoxetine, mianserin, buproprion, buproprion metabolite hydroxybuproprion, nomifensine and viloxazine (Vivalan®), especially a selective noradrenaline reuptake inhibitor such as reboxetine, in particular (S,S)-reboxetine; a dual serotonin-noradrenaline reuptake inhibitor, such as venlafaxine, venlafaxine metabolite O-desmethylvenlafaxine, clomipramine, clomipramine metabolite desmethylclomipramine, duloxetine, milnacipran and imipramine; an inducible nitric oxide synthase (iNOS) inhibitor such as S-[2-[(1-iminoethyl)amino]ethyl]-L-homocysteine, S-[2-[(1-iminoethyl)-amino]ethyl]-4,4-dioxo-L-cysteine, S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine, (2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)-butyl]thio]-5-chloro-3-pyridinecarbonitrile; 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-4-chlorobenzonitrile, (2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiazolebutanol, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl) butyl]thio]-6-(trifluoromethyl)-3 pyridinecarbonitrile, 2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonitrile, N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-carboxamidine, or guanidinoethyldisulfide; an acetylcholinesterase inhibitor such as donepezil; a prostaglandin E2 subtype 4 (EP4) antagonist such as N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl}amino)-carbonyl]-4-methylbenzene sulfonamide or 4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]benzoic acid; a leukotriene B4 antagonist; such as 1-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylic acid (CP-105696), 5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E-hexenyl]oxyphenoxy]-valeric acid (ONO-4057) or DPC-11870, a 5-lipoxygenase inhibitor, such as zileuton, 6-[(3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl])phenoxy-methyl]-1-methyl-2-quinolone (ZD-2138), or 2,3,5-trimethyl-6-(3-pyridylmethyl), 1,4-benzoquinone (CV-6504); a sodium channel blocker, such as lidocaine; or a 5-HT3 antagonist, such as ondansetron; and the pharmaceutically acceptable salts and solvates thereof.
 34. The method according to claim 30, wherein the nucleic acid molecule is administered in a composition comprising a pharmaceutically acceptable carrier and/or an excipient.
 35. A kit comprising: (a) the pharmaceutical composition according to claim 26; and (b) instructions for the administration of an effective amount of the pharmaceutical composition to an individual for the treatment of pain and/or a symptom of pain. 